Biomedical subjects

K Minoguchi

Publications and source records attributed to K Minoguchi.

30 records · Page 2Linked to original sources

PubMed1995

A 77-kDa protein associates with pp125FAK in mast cells and becomes tyrosine-phosphorylated by high affinity IgE receptor aggregation.

The focal adhesion kinase, pp125FAK, is a novel non-receptor protein tyrosine kinase expressed in different cells including mast cells. Here we report that a 77-kDa protein associates with pp125FAK in the mast cell analog, rat basophilic leukemia (RBL-2H3) cells. When pp125FAK immunoprecipitates were subjected to an in vitro kinase assay, there was prominent phosphorylation on tyrosine of pp125FAK and of a 77-kDa protein. By V8 protease digestion mapping and by immunoblotting with two different anti-pp125FAK antibodies, the 77-kDa protein was distinct from pp125FAK. This Fak Associated Protein or FAP was detected in RBL-2H3 cells but not in fibroblasts. The aggregation of the high affinity IgE receptor, Fc epsilon RI, induced the in vivo tyrosine phosphorylation of FAP. However, there was a marked decrease in the in vitro phosphorylation of FAP in the immunoprecipitates from Fc epsilon RI aggregated cells. Both of these Fc epsilon RI-mediated effects were enhanced by cell adhesion. There was strong association of FAP with non-tyrosine-phosphorylated pp125FAK. Thus this interaction does not appear to be mediated by the Src homology 2 domain. Together the data indicate that FAP associates with pp125FAK and suggest that FAP may play a role in Fc epsilon RI signaling.

3T3 Cells↗

PubMed1994

The aggregation of the high affinity IgE receptor induces tyrosine phosphorylation of paxillin, a focal adhesion protein.

Tyrosine phosphorylation of proteins is an essential component of high affinity IgE receptor (Fc epsilon RI) signaling and secretion. This signaling and secretion is also dependent on the organization of the cytoskeleton. Here we report that the aggregation of Fc epsilon RI on rat basophilic leukemia cells results in tyrosine phosphorylation of the cytoskeletal protein, paxillin. Tyrosine phosphorylation of paxillin is a relatively late event after Fc epsilon RI aggregation. Both the direct increase in intracellular Ca2+ with calcium ionophore and the activation of protein kinase C (PKC) with PMA induced tyrosine phosphorylation of paxillin. The optimal tyrosine phosphorylation of paxillin by Fc epsilon RI aggregation required PKC and extracellular Ca2+. However, there was also Fc epsilon RI-mediated tyrosine phosphorylation of paxillin independent of Ca2+ influx or PKC activation. By fluorescent microscopy, cell stimulation induced a redistribution of paxillin toward the periphery of the cells. Although Fc epsilon RI aggregation induced tyrosine phosphorylation of paxillin in nonadherent cells, adherence markedly enhanced this phosphorylation. Together, the data suggest a role for paxillin in Fc epsilon RI signaling.

Animals↗

PubMed1994

The anti-ganglioside monoclonal antibody AA4 induces protein tyrosine phosphorylations, but not degranulation, in rat basophilic leukemia cells.

The monoclonal antibody AA4 (mAb AA4) recognizes novel alpha-galactosyl derivatives of GD1b on rat basophilic leukemia (RBL-2H3) cells. The binding of mAb AA4 induced protein tyrosine phosphorylations without histamine release. Several of the same proteins including Fc epsilon RI beta, Fc epsilon RI gamma, p72syk, and phospholipase C-gamma 1 were tyrosine-phosphorylated by mAb AA4 binding and by the activation of the high affinity IgE receptor, Fc epsilon RI. There was also activation of the p53/56lyn and p72syk protein-tyrosine kinases, but compared to direct Fc epsilon RI activation, mAb AA4 did not result in increased tyrosine phosphorylation of pp105-115 or pp125FAK, and the receptor subunits (Fc epsilon RI beta and Fc epsilon RI gamma) were more heavily phosphorylated. Furthermore, the time course of the phosphorylations with mAb AA4 was slower than that induced by Fc epsilon RI aggregation. By immunofluorescence, the tyrosine-phosphorylated proteins after mAb AA4 stimulation were localized in patches at the cell membrane and in areas of cell-cell contact, whereas after Fc epsilon RI activation, there was a reticular cytoplasmic pattern. There were no protein tyrosine phosphorylations either when Fc epsilon RI was saturated with IgE or when F(ab')2 fragments of mAb AA4 were used, although the F(ab')2 fragments still induced morphological changes. There was also coprecipitation of the beta and gamma subunits of Fc epsilon RI with the anti-ganglioside antibody. These data strongly suggest the involvement of Fc epsilon RI in the antiganglioside-induced protein tyrosine phosphorylations. Moreover, phosphorylations of these proteins including the beta and gamma chains of Fc epsilon RI and activation of p53/56lyn and p72syk did not result in histamine release.

Animals↗

PubMed1994

Activation of protein tyrosine kinase p72syk by Fc epsilon RI aggregation in rat basophilic leukemia cells. p72syk is a minor component but the major protein tyrosine kinase of pp72.

Aggregation of the high affinity IgE receptors (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in protein tyrosine phosphorylations. Previously we reported that there is prominent tyrosine phosphorylation of approximately 72-kDa proteins (pp72) and that the tyrosine kinase p72syk is one component of pp72. Here we studied further the relationship of p72syk to pp72. The aggregation of Fc epsilon RI induced the activation of p72syk which was parallel to its tyrosine phosphorylation. By in vitro kinase assay of immune complexes purified with anti-phosphotyrosine antibodies, p72syk was the major pp72 tyrosine kinase. However, by immunoblotting with anti-phosphotyrosine antibodies, p72syk was a minor component of pp72. The heterogeneous nature of pp72 was indicated by different studies. Under optimum conditions of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pp72 consisted of a heterogeneous group of 69-, 71-, and 72-kDa tyrosine-phosphorylated proteins. There were differences in the tyrosine phosphorylation of these proteins in cells activated in the absence of extracellular calcium or when stimulation was with the calcium ionophore A23187 or with phorbol myristate acetate. One of the proteins migrating at 69 kDa was p72syk. By two-dimensional gel electrophoresis pp72 was found to consist of multiple tyrosine-phosphorylated protens including 71-80-kDa proteins that associate with p53/56lyn. A 75-kDa tyrosine-phosphorylated protein, different from pp72, was identified as p75HS1 (SPY75). These results demonstrate the heterogeneous nature of the pp72 and that p72syk is activated after Fc epsilon RI aggregation.

Amino Acid Sequence↗

PubMed1994

Src family tyrosine kinase p53/56lyn, a serine kinase and Fc epsilon RI associate with alpha-galactosyl derivatives of ganglioside GD1b in rat basophilic leukemia RBL-2H3 cells.

The monoclonal antibody (mAb) AA4 recognizes two alpha-galactosyl derivatives of the GD1b ganglioside on rat mast cells and on the rat basophilic leukemia RBL-2H3 cultured cell line. Here we demonstrate that mAb AA4 coprecipitated both protein tyrosine and serine kinases. In contrast, a monoclonal antibody to the GD3 ganglioside did not coprecipitate any kinase activity. In kinase assays of mAb AA4 immunoprecipitates there were phosphorylated proteins of 71-80, 53/56, and 41/42 kDa. All proteins were phosphorylated on tyrosine, whereas the 71-80- and 41/42-kDa proteins were also phosphorylated on serine residues. The precipitation of these proteins by mAb AA4 correlated with the presence of the alpha-galactosyl derivatives of GD1b. The 53/56-kDa proteins were identified as the Src-related tyrosine kinase p53/56lyn. The presence of p53/56lyn in the mAb AA4 immunoprecipitates was specific and was observed when several different detergents were used. The same 71-80-kDa tyrosine-phosphorylated proteins were immunoprecipitated by mAb AA4 and anti-Lyn antibodies and may play a role in the interaction of p53/56lyn with the gangliosides. Although there is a weak association of the high affinity IgE receptor with these gangliosides, the coprecipitation of p53/56lyn with mAb AA4 was not secondary to the association of this kinase with receptor. These complexes of gangliosides and several proteins that include p53/56lyn, a serine kinase, and the high affinity IgE receptor could play an important role in receptor-mediated signal transduction.

Animals↗

PubMed1994

Src family tyrosine kinase Lyn binds several proteins including paxillin in rat basophilic leukemia cells.

Aggregation of the high affinity IgE receptors on rat basophilic leukemia (RBL-2H3) cells results in protein tyrosine phosphorylation although the receptor has no intrinsic enzymatic activity. The Src related protein tyrosine kinase p53/56lyn present in RBL-2H3 cells could play a role in this reaction. Here we have isolated the cDNA for rat Lyn and found it to be very homologous at the amino acid level to both the human and mouse proteins. A bacterially expressed maltose binding protein-Lyn (MBP-Lyn) fusion protein was already tyrosine phosphorylated and had tyrosine kinase activity. In a filter-binding assay, MBP-Lyn fusion protein (at 0.1 microM) specifically bound to several proteins of RBL-2H3 cells. In lysates of IgE receptor-activated cells, there was increased binding of MBP-Lyn to 65, 72, 78 and 110 kDa tyrosine phosphorylated proteins. The 72, 78 and 110 kDa tyrosine phosphorylated proteins were precipitated by a fusion protein containing the Lyn Src Homology 2 (SH2) domain. The 72 kDa Lyn binding protein was different from p72syk. Furthermore, paxillin, a cytoskeletal protein, was identified as one of the Lyn binding proteins. Thus Fc epsilon RI mediated signal transduction in RBL-2H3 cells may result from the interaction of p53/56lyn with paxillin, pp72, pp110 and other proteins.

Amino Acid Sequence↗

PubMed1993

Change in responsiveness of airway and beta-adrenoceptor in guinea pigs.

Allergen inhalation challenge resulted in significant (p < 0.05) increase in airway responsiveness to methacholine soon after immediate airway response (IAR) in guinea pigs actively sensitized with inhaled ovalbumin (OA) in vivo. We have investigated the involvement of thromboxane (Tx) A2 and platelet activating factor (PAF) in this airway hyperresponsiveness (AH). Pretreatment with CS-518, a selective thromboxane synthetase inhibitor, significantly inhibited both IAR (p < 0.07) and AH (p < 0.01), while pretreatment with WEB 2086, a PAF receptor antagonist, significantly inhibited only IAR (p < 0.05), but not AH after IAR. Propranolol, when inhaled before OA exposure, had no effect on bronchomotor tone, but if inhaled after IAR, it caused immediate death of guinea pigs. This result suggests that hyperresponsiveness of beta-adrenoceptor to propranolol may be induced by IAR. Examination of bronchoalveolar lavage (BAL) fluid revealed that no changes in cellular content were observed 60 min after OA exposure. In vitro, responsiveness of tracheal smooth muscle to carbachol was not changed by sensitization. Furthermore, anaphylactic reaction in vitro had no effect on the responsiveness. We conclude that airway responsiveness increases soon after IAR when infiltration of inflammatory cells is not yet found in vivo. It is also suggested that TxA2 but not PAF is involved in AH. Hyperresponsiveness to propranolol after IAR in OA sensitized guinea pigs illustrates the possibility of changes in function of beta-adrenoceptor after IAR.

Animals↗

PubMed1991

[The mechanism of airway hyperresponsiveness following immediate bronchial response in ovalbumin (OA)-sensitized guinea pigs].

We have previously demonstrated that airway responsiveness was enhanced following a late bronchial response (LBR) after an allergen challenge in ovalbumin (OA)-sensitized guinea pigs. The purpose of the present studies was to evaluate whether airway responsiveness to methacholine increased after an immediate bronchial response (IBR) and the possible involvement of the beta-adrenoceptor dysfunction in OA-sensitized guinea pigs. Guinea pigs were actively sensitized by aerosolized OA. Following OA exposure, IBR appeared. After IBR when specific airway resistance returned to the base line value, airway responsiveness to methacholine increased significantly. Before OA exposure, propranolol induced bronchoconstriction (PIB) was not provoked, however, after IBR, PIB was provoked and the guinea pigs died because of severe bronchoconstriction. These results suggest that airway responsiveness to methacholine increases significantly after IBR. Furthermore, the dysfunction of the beta-adrenoceptor may be a mechanism of this hyperresponsiveness in OA-sensitized guinea pigs.

Animals↗

PubMed1990

[Histologic characterization of late asthmatic response in guinea pigs].

We examined lung tissues in a guinea pig model actively sensitized by inhalation of aerosolized ovalbumin (OA) and found reproducible late bronchial responses (LBR) 7 h and 24 h after OA challenge. Light microscopic examination revealed bronchoconstriction, damage to the epithelium, eosinophil infiltration in both the epithelium and subepithelium, retention of mucous in the pulmonary bronchial lumens and a decrease in the mucous content of Periodic Acid-Schiff (PAS) stain positive cells in the bronchial epithelium in LBR. An increase in the number of PAS-stain positive cells of in the bronchial epithelium was also observed in LBR. Electron microscopic examination revealed eosinophil migration around mast cell at 7 hs after OA challenge. Two different mechanisms of degranulation of eosinophil specific granules were observed in LBR. In one mechanism, the granule core changes after its matrix has changed. In the other mechanism, the granules were exocytosed as a whole, accompanied by the cell membrane and densely clumped chromatin, suggesting burst of eosinophil.

Animals↗

PubMedDate not supplied

IL-10 induces a Th2 cell tolerance in allergic asthma.

BACKGROUND: Interleukin (IL)-10 induces a long-term antigen-specific anergy in human CD4+ T cells. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from house dust mite (Dermatophagoides farinae, Der f)-sensitized asthmatic patients. PBMCs were stimulated with Der f antigen for 72 h immediately after purification or after 48 h of resting culture with medium, and IL-10 and IL-5 in the culture supernatant were measured. PBMCs were also stimulated with Der f antigen for 7 days either immediately after purification or after 48 h of resting culture, after which cells were collected. Secondary proliferative responses of these cells to stimulation for 3 days with Der f antigen and mitomycin C-treated PBMCs as antigen-presenting cells or with phorbol myristate acetate (PMA) plus calcium ionophore were investigated. RESULTS: Stimulation of PBMCs with Der f antigen immediately after purification significantly increased the proliferative response and IL-5 production. Stimulation of PBMCs with Der f antigen after resting culture with medium alone for 48 h significantly decreased IL-5 production and markedly increased IL-10 production. Although activation of cells with Der f antigen immediately after purification significantly increased secondary proliferative responses, stimulation after 48 h of resting culture failed to increase secondary proliferative responses. However, proliferation recovered when cells were activated with PMA plus calcium ionophore. CONCLUSION: These results suggest that antigen-specific Th2 cells are anergized by IL-10 and that Th2 cell tolerance may suppress eosinophilic inflammation in allergic asthma.

Allergens↗

PubMedDate not supplied

Step-down and step-up therapy in moderate persistent asthma.

BACKGROUND: A step-down therapy may be more beneficial for the management of asthma than a step-up therapy. METHODS: Eighty-two asthmatic patients with moderate persistent asthma were enrolled in the study and randomized into three groups. One group of patients received 400 microg/day of beclomethasone dipropionate (BDP) for 4 weeks and then 800 microg/day for another 4 weeks (step-up group). The other two groups of patients received 1,200 microg/day of BDP for 4 weeks with or without short-term oral steroid (prednisolone, 0.5 mg/day for 1 week) and then 800 microg/day for another 4 weeks (step-down group). Severe exacerbation of asthma, asthma symptoms, respiratory function and rescue use of inhaled beta(2)-agonists were monitored. If asthma was well controlled, the dose of BDP was decreased every 3 months and if asthma was exacerbated, the dose of BDP was increased until 8 months after the initial treatment. RESULTS: Twenty-two patients during the run-in period, 4 patients in the step-up group, 2 patients in the step-down group treated with a high dose of BDP and no patients in the step-down group with oral steroids during first 4 weeks dropped out because of severe exacerbation of asthma. Although asthma symptoms and respiratory function significantly improved 8 weeks after the therapy in all groups, more significant and prompt improvements of these parameters were observed in patients of the step-down group than in patients of the step-up group after the first 2 weeks of treatment. Furthermore, step-down therapy with short-term oral steroid resulted in the lowest maintenance doses of BDP at 8 months of the three groups. CONCLUSIONS: These results suggest that step-down therapy starting with a high dose of inhaled steroid and short-term oral steroid is more effective in gaining prompt control of asthma and reducing the severe exacerbation of asthma and the maintenance dose of inhaled steroids than a step-up therapy starting with a low dose of inhaled steroids in patients with moderate persistent asthma.

Administration, Inhalation↗

PubMedDate not supplied

The mechanism of airway hyperresponsiveness following immediate asthmatic response in ovalbumin-sensitized guinea-pigs.

The mechanism of airway hyperresponsiveness (AH) following immediate asthmatic response (IAR) was investigated in ovalbumin (OA)-sensitized guinea-pigs. After IAR, airway responsiveness was significantly increased and propranolol-induced bronchoconstriction (PIB) was so severe that guinea-pigs died. A thromboxane (Tx) synthetase inhibitor CS-518 significantly inhibited AH and PIB, whereas a PAF receptor antagonist WEB-2086 was only slightly effective. These results suggest that TxA2 plays an important role and PAF a partial role in AH following IAR.

Animals↗